Method for diagnosing neuronal diseases and for treating primary hemostasis deficiency

ABSTRACT

Pharmaceutical compositions for treatment of primary hemostasis deficiency and methods of treatment of primary hemostasis deficiency are disclosed. Also, methods for diagnosing neuronal diseases and for suppressing the immune system are disclosed. Isoforms responsible for maintaining primary hemostasis, T-cell mediated immune responses, neuron specific TPH, and a neuronal tryptophan hydroxylase are disclosed.

This application is a 371 of PCT/DE01/03178 filed on Aug. 27, 2001, and claims benefit of Germany 10043/24,0 filed on Aug. 31, 2000.

BACKGROUND OF THE INVENTION

The present invention relates to a method for diagnosing neuronal diseases and or treating primary hemostasis deficiency. The invention further relates to a method for suppressing the immune system, which is inter alia significant for transplantation medicine and for the treatment of allergies. The invention is used in the field of medicine and pharmaceutical industry.

Several behavioral disorders, such as depressions, alcoholism, drug abuse, isomnia and nutritional disturbances, are disorders tightly connected to the central nervous system. The tissue hormone serotonin plays an essential role at many levels. Serotonin is a neurotransmitter inter alia for the stimulation of the peristalsis, vasodilatation and constriction (dosage depending), respectively, and the increase of the tonicity in the respiratory tract. It is not only a neurotransmitter in the CNS, but, in addition, a compound that can be found everywhere in the periphery, where serotonin was first discovered due to its activity as strong vascular constrictor (Rapport et al., J. Biol. Chem. 176:1237, 1948).

In addition, serotonin plays an essential role in primary hemostasis, that is, the inhibition of bleedings. The serotonin that is stored in the platelets is excreted at sites of vascular lesion, whereupon it interferes with the primary hemostasis (Holland, Proc. Soc. Exp. Biol. Med. 151:32-39, 1976).

Furthermore, the serotonin of the platelets further mediates between components of the immune system (Geba et al., J. Immmunol. 157:557–565, 1996). The mechanisms that thereby take place are yet unknown.

It is known that the enzyme tryptophan-hydroxylase (TPH), an enzyme that is expressed in neurons and pheripheric tissue, catalyses the yield-limiting step in the biosynthesis of serotonin, and is therefore essential for the function of the serotonergic system in the CNS and the periphery (Boadle-Biber, Prog. Biophys. Mol. Bio. 60:1–15, 1993).

There are already some medical applications, which rely on the effect of serotonin in the body. The increased release of serotonin by Rauwolfia-Alkoloides, and the retardation of the serotonin decay using MAO (Monoaminooxidase) inhibitors, respectively are used for the treatment of mental depressions, while its antagonist, methysergide, find its application in the treatment of migraine.

It has not yet been achieved to control the serotonin production in the body in such a manner that an effective treatment of the above-mentioned and other pathological phenomena are possible.

It is therefore an object of the invention to improve the diagnosis of neuronal diseases of the above-mentioned kind. It is a further or alternative object to develop agents for the treatment of the primary hemostasis deficiency as well as agents for suppressing the immune system based on new findings of the effect of serotonin.

SUMMARY OF THE INVENTION

The invention is based on the essential discovery that serotonin is synthesized in the body by TPH isoenzymes that are differently expressed in the neurons and the peripheric tissues. Gene targeting was used to show that an isoform, the peripheral enzyme (referred to in the following as TPH), is responsible for maintaining the primary hemostasis and T-cell mediated immune responses. Another isoform, the newly identified neuron specific TPH (referred to as nTPH) synthesizes serotonin independently thereof in the CNS.

Furthermore, some mechanisms could be elucidated. It was found

-   a) that the major step of serotonin in the primary hemostasis is     mediated by the von Willebrand-factor. Furthermore, it was     established that -   b) the serotonin in the platelets is essential for the normal number     of circulating CD4⁺ cells, as well as for their normal activity, and     thus provides a new target for an immune suppressive treatment.

In summary, it can be established that two separately regulated serotonergic systems exist in the CNS and the periphery which are defined by the peripheral isoform TPH and the neuronal isoform nTPH, respectively. This result is an important contribution for the use of these systems within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the production of TPH(−/−)-mice.

FIG. 2 shows the quantification of tryptophan (Trp) and serotonin (5-HT) in peripheral tissues and of Trp, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in selected areas of the brain.

FIG. 3 shows disturbed hemostasis in TPH(−/−)-mice.

FIG. 4 shows disturbed T-cell mediated immune responses in TPH(−/−)-mice.

FIG. 5 illustrates the complexity of TPH-expression in a mouse.

FIG. 6 shows the characterization of a new exon 2b in mice.

FIG. 7 is a schematic representation of TPH gene structure.

FIG. 8 shows the amino acid-sequence alignment of a new regulatory domain of TPH with other AAAHs.

FIG. 9 shows the amino acid sequence of nTPH.

DETAILED DESCRIPTION

The dichotomy of the serotonin-biosynthesis on the molecular level results in unexpected diagnostic and therapeutic possibilities. This relates to correlations between peripheric and central nervous serotonergic metabolites. In addition, this relates to possibilities of diagnostic fine-correlations, since the subtractive determination of the concentrations of the metabolites as present allows for the ascertainment of the CNS metabolites in the periphery. The peripheral serotonin-biosynthesis can thus transiently be inhibited specifically in order to subsequently determine the peripheral metabolite concentrations that stem from the CNS. The advantages of such a diagnostic method, in particular in the case of persisting psychiatric disorders, should outbalance the transient risk of bleeding episodes and distorted cell-mediated immune responses, since an improved therapy can follow after a more precise diagnosis.

The invention primarily relates to inhibitors or promoters, respectively of both TPH-isoforms which can be used for diagnosing neuronal diseases and for treating primary hemostasis deficiency. Furthermore, the invention relates to the use of these inhibitors for suppressing the immune system, which is of importance inter alia for transplantation medicine and for the treatment of allergies.

The method is characterized by a modulation of the regulation of nTPH and/or TPH and thus the specific serotonin production in the body. It was shown that the peripheral TPH is essential for primary hemostasis and for the T-cell-mediated immune response, whereas the newly identified nTPH, which is independently expressed and synthesizes serotonin in the CNS is responsible for the serotonergic effects in behavioral physiology. This results in new pharmacological possibilities for immune suppression and for the therapeutic manipulation of hemostasis.

The modulation of the nTPH- and/or TPH-regulation takes place in the following ways:

Specific inhibitors of the peripheral TPH-isoforms were developed that rely on the molecular differences of the TPH-isoforms as described, and also inhibitors that cannot pass the blood-brain-barrier. Specific inhibitors for the TPH-isoforms can be found in vitro using fairly simple screening methods, since the cDNAs of the two isoforms that were obtained can be used for the specific expression of the pure isoforms using expression systems. These inhibitors, in addition to a diagnostic use, account also for therapeutical uses, since increased serotonin levels in the blood are found in a plurality of complications.

a) In this context it is worth mentioning that during the use of SSRI (serotonin specific reuptake inhibitors)-antidepressive agents in the psychopharmacological therapy, as side effects in some cases serious bleeding complications became known (Goldberg, Arch. Fam. Med. 7:78–84, 1998). These side effects can be traced back to the decreased serotonin-level in platelets due to said therapy (Bottlender et al., Fortschr. Neurol. Psychiatr. 66:32–35, 1998). Also associated with this field of application are the new findings of the participation of serotonin in primary hemostasis. One side effect of antidepressive agents which act in a blocking fashion on the serotonin-transporter is the incidence of acute bleeding episodes, apparently due to the insufficient serotonin storage in the thrombocytes of the patients that are treated. That is to say that the serotonin-transporter of the thrombocytes is identical with the specific target of the antidepressive agents, the serotonin-transporter of the CNS. In the case of such bleeding episodes the antidepressants are usually displaced, which results in a decline of the status of the afflicted patients and an increase of the depression-induced risk for suicide. This can be avoided if commercially available von Willebrand-factor (vWF) is infused during such an acute bleeding episode.

b) Increased serotonin-concentrations during the pre-eclampsia correlate with the increase of the CD4⁺-T-cell-subpopulations. Using specific inhibitors of the peripheral TPH-isoform, an early treatment of the imminent pre-eclampsia is enabled, which neither exerts an influence on the serotonin-biosynthesis in the maternal nor in the fetal brain.

c) In addition to these applications, a positive influence of only peripherally acting inhibitors on transplantation medicine also can be achieved. An essential limitation in a common immune suppressive therapy is given by the nephro-, hepato- and neurotoxic side effects of the most commonly used substance clyclosporin A (CsA). More recent immune suppressants of natural origin, such as FK506 and rapamycin in combination preparations together with CsA thus allow a more effective (additive) immune suppresive treatment having lower side effects, since CsA is thus applied at a lower dosage.

The immune suppressive effect of lowered serotonin levels in the blood can be used in order to apply toxic substances, CsA, FK 506, and rapamycin, at a lower dose.

According to the invention, the manipulation of the nTPH- and/or TPH-regulation can take place in the following ways:

The specific down-regulation of nTPH and/or pTPH using molecular biology takes place with ribozymes, antisense-oligonucleotides or by antisense-RNA-expression, wherein the differences in the sequences of the isoform-mRNAs allow for the modulation of only one of each mRNA at once. In addition, according to the invention, also pharmacological preparations are employed using specific TPH-inhibitors, such as, for example, p-chlorophenylalanine or p-ethynylphenylalanine.

The serotonin production is preferably stimulated by tissue specific over-expression of the TPH-isoforms using molecular biology. Pharmacologically, for example, the precursor-compound 5-hydroxytryptophan, and also substituted analogues, and, last but not least, also serotonin itself can be administered.

The method according to the present invention for diagnosing neuronal diseases is characterized in that a specific inhibition of the peripheral serotonin-biosynthesis is performed, subsequently the metabolite concentrations derived from the CNS are determined, and based on a comparative curve the grade of the disease is ascertained. For this, it is required to use compounds that cannot pass the blood-brain-barrier.

The invention further relates to the newly identified neuronal tryptophan-hydroxylase (nTPH) that differs from the known TPH its regulatory domain (the catalytic domain is identical). nTPH has the amino acid sequence, that is depicted in FIG. 9.

The invention shall be described in detail in the following based on embodiments (figures), in which:

FIG. 1. Production of TPH(−/−)-mice

-   A. Schematic representation of the targeting-method. The first four     of the 11 exons of the TPH-gene are depicted. The knock-out     construct was integrated by homologous recombination in one allele     of the ES-cells, whereby the first coding exon of the TPH-gene was     inactivated. Furthermore, the integrated neomycin     resistance-cassette contained a transcriptional-stop-sequence. The     positions of the analytic amplicons are depicted for wild-type and     knock-out-indentification, as well as the positions of the internal     and external Southern blot probes. Identical Eco-RI-sites that     enable the identification of wild-type and knock-out-animals in     Southern blots are depicted in bold (RI). Restriction sites: RI: Eco     RI; Hd: Hind III; Bm: Bam HI; Sc:Sac I. -   B. Agarose gel-electrophoresis of analytical PCR-products. As stated     in A, a 1.1 kb-fragment for the wild-type-allele, and a 1.3     kb-fragment for the knock-out-allele is obtained, respectively. -   C. Southern blot-examination of Eco RI-digested genomic DNA. As     stated in A, both probes detect a 10 kb-fragment for the wild-type     allele and an 8 kb-fragment for the knock-out-allele.

FIG. 2. Quantification of trypthophan (Trp) and serotonin (5-HT) in peripheral tissues and of Trp, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in selected areas of the brain.

-   A. Trp and 5-HT in whole blood and in duodenum-samples of 129SvJ-,     C57BL/6-, TPH(−/−)-, and TPH(+/+)-mice. N.d: not detectable (<25     fg/μl). *: statistically significant compared to all other     mice-straines (p<0.05). -   B. Trp, 5-HT and 5-HIAA in hippocampus- and frontal cortex-samples     of 129SvJ-, C57BL/6-, TPH(−/−)-, and TPH(+/+)-mice. *: statistically     significant compared to all other mice-strains (p<0.05). 5-HT is     significantly lowered in the hippocampus of TPH(−/−)-mice compared     with TPH(+/+)-mice, nevertheless not compared with the other     laboratory-mice-strains.

FIG. 3. Disturbed hemostasis in TPH(−/−)-mice.

-   A. Prolonged bleeding times of TPH(−/−)-animals. Normal bleeding     times of control-animals are at 8-11 min, whereas TPH(−/−)-mice     bleed at an average of 35 min. -   B. Healing of the phenotype by intracardial 5-HT-injection. The     5-HT-amounts applied result in 1/10 and the ten-fold of the normal     blood concentrations. -   C. Healing of the phenotype by intracardial von Willebrand-factor     injection. The effect is specific for the TPH(−/−)-mice, whereas     control-animals are not affected.

FIG. 4. Disturbed T-cell-mediated immune responses in TPH(−/−)-mice.

-   A. Time course of the net swelling of ears after oxazolone     treatment, indicative for T-cell-mediated hypersensitivity of the     late type. TPH(−/−)-mice do not exhibit an allergic swelling     reaction. -   B. Determination of leucocyte-subpopulations in the peripheral blood     of TPH(−/−)-animals using flow-cytometry. Only CD+4-T-cells     (CD4+/CD3+) are significantly reduced (p<0.05), but not     CD8+-T-cells. -   C. Exemplary illustration of the rejection of     skin-second-order-transplants. Arrowheads indicate the sensitizing     transplants of the first order, arrows the transplants of the second     order. In comparison with the well-healing transplants on     TPH(−/−)-animals, the transplants on TPH(+/+)- and C57BL/6-animals     can at the outer edges of wounds (white rejection) be identified by     insufficient revascularization and infections at the same time or     are already completely rejected (transplant of first order and two     of the transplants of second order on the C57BL/6-animal).

FIG. 5. Complexity of TPH-expression in the mouse.

-   A. Sequence alignment of the published cDNA (Stoll et al., 1990) of     the cloned alternatively spliced cDNA having a 34 bp-elongation of     exon 3 and genomic DNA. Chromosomal DNA has the 34 bp-elongation     directly after the splicing-donor-site of exon 3, whereupon an     obligatory GT-dinuclotide follows; the elongated exon 3 is in the     following designated exon 3b. -   B. RNase protection assays of 80 μg total-RNA of the organs as     indicated and 10 μg of the mastocytoma-cells P815 (2x) with the cDNA     described in 5A. The probe clearly identifies six splice variants of     the mouse-TPH-mRNA. It has to be noted that only a set of three     variants is expressed in the TPH-KO-mice. The weak band at the     position of the known TPH-mRNA that can be seen in the duodenum and     lung of TPH-KO(−/−)-animals results from a high sequence homology of     the variants. The P815-RNAs show how comparatively low the     expression of TPH is in tissues, namely nearly at the level of     detectability. -   C. Alternative splicing between exon 3 and exon 4. The     splicing-donor-(SDS)- and splicing-acceptor-sites (SAS) that are     involved are indicated. The parts of the sequence of the published     sequence are marked as exon 3a and exon 4a. Splicing of SDS2 on SAS     1 results in the C-variants, which only code for the regulatory     domain of the TPH-isoforms (cf. FIG. 7B). The splicing of SDS2 on     the internal SAS2 of exon 4 eliminates the frame shift that would be     the result of the additional 34 bp of exon 3b. The constant regions     are shaded in light grey, the alternative segments in dark grey.     Furthermore, the alternative segments as well as flanking     intron-sequences are indicated in italic.

FIG. 6. Characterization of the new exon 2b

-   A. Genomic sequence of exon 2b of the mouse with flanking sequences.     The ORF in the trinucleotide-notation is marked in bold. In the     5′-flanking region, a part of the putative new promoter is shown,     wherein one TATA-box and the most probable start of transcription     (+1) are indicated. In the 3′-region, the splicing-donor-site of     exon 2b is depicted. Intron-sequences have been indicated in small     letters in italic, wherein the short intron of 53 bp was     symbolically bridged from the segments of the ORF using a line. For     clarity, the respective consensus-sequences are indicated below the     splicing-donor-sites,-branching-sites, and -accepting-sites,     respectively that are involved. -   B. Examination of the exon-intron-structure of the new exon 2. Five     out of eight nucleotides in the mouse are identical with the     consensus-sequence of splicing-donor-sites (bold and underlined),     four in the rat and three in human. The intron between the new exon     2 and exon 3 has an exact length of 948 bp in the mouse, similarly     as in rat and in human.

FIG. 7. Schematic depiction of the TPH-gene-structure

-   A. Schematic comparision of the known splicing-structure (top) with     the structure that was obtained in the present work for the gene     segments of the regulatory domain (bottom). The promoter position     that is supported by experimental evidence, was marked with P2. -   B. Assembly of all newly discovered mRNA-variants. The promoter 1 is     primarily active in the periphery, whereas the new promoter 2 is     responsible for expression in CNS.

The mRNA that is indicated as variant A is the known form of the TPH-mRNA. The variant A′ was already cloned and codes for an active TPH-Isoform. The variants B and B′ are not yet cloned, nevertheless, they have been clearly identified for several times now using RPA-assays. Variants C and C′ lead to the expression of only the regulatory domains. For clarity, the mRNAs are only depicted up to the 6^(th) exon of the overall 11 exons.

FIG. 8. Amino acid-sequence-alignment of the new regulatory domain of TPH with other AAAHs

The upper part of the sequences shows the new segment of TPH. Below, the conserved parts of the sequence of the regulatory domains of the AAAHs are indicated. The sequences have each been aligned with another using the clustal W-program and have been assembled manually. In doing so, larger gaps were partially allowed in order to indicate the identical structural elements. Noticeable sequence motifs are framed and individually named. Four of the five motifs are identical with elements of the secondary structure of the PAH. Overlapping in part with the IEDN-motif, a region of the regulatory domain is framed and shaded in gray that reaches into the groove of the catalytic centre, as it is known from christallographic examinations of the PAH. Further explanations in the text. The sequences used are: nstph=neuron-specific TPH (the new variant) of the mouse, musth=TH of the mouse, muspah and humpah=murine and human PAH, ptph=peripheral TPH of the mouse (known variant).

FIG. 9. Amino acid-sequence of the nTPH 

1. A pharmaceutical composition for the treatment of primary hemostasis deficiency, comprising: an agent that acts on the level of serotonin in the blood selected from the group consisting of p-chlorophenylalanine, p-ethynylphenylalanine, serotonin, 5-hydroxyindoleacetic acid (5-HIAA), 5-hyroxytryptophan, substituted analogues of 5-hydroxytryptophan, von Willebrand-factor and combinations thereof, wherein said composition further comprises a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier or combinations thereof.
 2. The pharmaceutical composition as recited in claim 1, further comprising an antidepressive agent that acts on the level of serotonin in the body.
 3. The pharmaceutical composition as recited in claim 1, wherein the agent that acts on the level of serotonin in the blood can not pass through the blood-brain barrier.
 4. The pharmaceutical composition as recited in claim 1, wherein the agent that acts on the level of serotonin in the blood is selected from the group consisting of 5-hydroxytryptophan, substituted analogues of 5-hydroxytryptophan, serotonin, von Willebrand-factor (vWF) and combinations thereof.
 5. A method for treatment of primary hemostasis deficiency in a mammal in need of treatment, comprising: administering to the mammal in need of treatment an effective amount of a pharmaceutical composition as recited in claim
 1. 6. The method for treatment of primary hemostasis deficiency in a mammal in need of treatment as recited in claim 5, wherein the mammal is a human.
 7. A method for treatment of a depressive disorder in a mammal in need of treatment, comprising administering to the mammal in need of treatment an effective amount of a pharmaceutical composition as recited in claim
 2. 8. The method for the treatment of a depressive disorder in a mammal as recited in claim 7, wherein the mammal is a human. 